The research of the Hematopoiesis Section is focused on the basic biology of stem cells and the use of stem cells as vehicles for cell and gene therapy. Hematopoietic stem cells (HSC) are a rare population of self- renewing cells that give rise to all cells in the peripheral blood, making them ideal vehicles for gene replacement therapy of inherited hematopoietic diseases. In addition, cells highly enriched for HSC can generate cardiac myocytes when injected into the healthy tissue surrounding a myocardial infarct or when mobilized into the peripheral blood with cytokines (e.g. G-CSF and SCF). Project 1: Biology of Hematopoietic Stem Cells Specific Aim 1.1: We hypothesized that the same hematopoietic stem cells that repopulated the bone marrow were responsible for the regeneration of cardiac myocytes after experimental myocardial infraction (MI). To test this hypothesis, we transplanted mice with bone marrow cells marked with a retrovirus vector containing the GFP gene. These mice then underwent a coronary artery ligation and stem and progenitor cells were mobilized into the peripheral blood with G-CSF and SCF. The infarct region was repopulated with diploid cells that contain the identical retrovirus markers found in peripheral blood cells.We conclude that the new cardiac myocytes are the progeny of cells that also give rise to peripheral blood cells. Specific Aim 1.2: We hypothesize that specific genes expressed in both HSC and stem cells isolated from skeletal muscle are responsible for maintaining an undifferentiated state. To test this hypothesis, we have isolated transcripts specifically expressed in HSC and in side population stem cells from mouse skeletal muscle cultures by cDNA. We have shown that Hmgb3 is a protein required to block differentiation of HSC and promote HSC self renewal. We will be looking for the protein and DNA targest of Hmgb3 in the coming year. The muscle stem cell transcripts will be compared to those we have identified previously in a cDNA subtraction library of HSC. We will select transcripts common to both libraries for analysis in transgenic mice. Gene transfer to HSC has recently been shown to cure Severe Combined Immune Deficiency, demonstrating that HSC gene therapy could be applied to more common diseases. We would like to develop a gene therapy for Sickle Cell Disease. However, current levels of gene transfer to HSC are too low to treat this disease. We have found that one important reason that gene transfer is so low is that the conventional retrovirus receptors on HSC are nearly undetectable. A second problem has been the instability of retrovirus vectors containing globin genes. Project 2: Gene therapy for the hemoglobinopathies Specific aim 2.1: We have shown that the receptors of the RD114 and FeLV-C retrovirus are expressed at high levels on hematopoietic stem cells, and that this leads to improved gene transfer to human hematopoietic cells in the sheep xenograft model. We are expanding these observations into marrow from patients with Lymphocyte Adhesion Deficiency and the hemoglobinopathies. Specific Aim 2.2: We hypothesize that stable retrovirus vectors containing globin genes linked to the promoters of genes expressed in erythroid cells can be generated that will allow expression of globin mRNA at levels adequate to treat Sickle Cell Disease and b-thalassemia. Our evaluation of the relative level of expression of red cell gene promoters using a transgenic mouse assay has shown that the AE-1 promoter linked to a chicken insulator element directs position independent, uniform, high-level, and copy number dependent expression. We have generated stable retroviruses with this construct which will be evaluated in the mouse b-thalassemia model. We have also demonstrated a more compact insulator element in the ankyrin that should lead to higher virus titers. We are currently evaluating the regulatory regions of the alpha spectrin, AHSP and AE-1 genes for other insulator elements. In related work we are evaluating the ability of the zeta globin chain to inhibit the polymerization of HbS in the mouse model.